Project abstract/summary Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly, and patients with the most common dry form suffer from limited treatment options. In dry AMD, some retinal pigment epithelial (RPE) cells undergo epithelial to mesenchymal transition (EMT) to survive microenvironmental stress, and can reside alongside both normal and dying RPE, contributing to the functional and morphological RPE heterogeneity that defines AMD. In humans and mice, mitochondrial and stress response defects have emerged as independent mechanisms to explain RPE abnormalities. In pilot experiments, knockdown of Pink1, the driver of mitophagy, causes death resistant EMT in human RPE cell lines in an Nrf2 dependent manner. Four cell types generated by knockdown of Pink1 and/or Nrf2 (normal, single knockdowns, and the double knockdown) show differences in morphology and viability, which simulates RPE heterogeneity. The central hypothesis to be tested in this proposal is that impaired mitophagy induces EMT in RPE cells through Nrf2 dependent retrograde signaling, from the mitochondria to the nucleus, and that failure of Pink1 mediated mitophagy and/or Nrf2 signaling contributes to RPE heterogeneity in dry AMD. The specific aims are: 1) Determine the extent that impaired mitophagy induces EMT in RPE cells. 2) Determine how Nrf2 dependent mitochondrial retrograde signaling induces a pro-survival EMT transcriptome in the RPE. 3) Determine the degree that mitophagy and mitochondrial retrograde signaling contribute to RPE heterogeneity with an AMD phenotype. The significance of this project is that successful completion will identify novel drug target pathways and that death resistant EMT cells can be targeted for therapeutic rejuvenation rather than cells committed to death. This study is innovative because it proposes i) that impaired mitophagy in conjunction with Nrf2 signalling is a trigger for EMT, ii) that the interplay of mitophagy and Nrf2 signalling can explain RPE heterogeneity, and iii) to use state-of-the art genetically modified human iPS cells generated by Crispr/Cas9 editing and unique mouse models. This project will take advantage of the PI's background in mitochondrial biology and molecular genetics and the PI will be further trained in retinal biology and animal models of AMD in Dr. Handa's lab (mentor), in stem cell editing by Dr. Zack (co-mentor) and in advanced cell biology by Dr. Sesaki (co-mentor), who are all leaders in their fields. The project is on track because of successfully established stem cell biology and animal facilities in the mentor's and co-mentors' labs, and the mouse strains for this study have already been generated. We have obtained Pink1 knockout stem cell line from our collaborator Dr. Dawson for Aim 1, and a CRISPR edited Nrf2 mutant is underway for aims 2 and 3. The PI is in the ideal environment for the proposed research as Wilmer has world- class facilities to complement the renowned scientists available within the entire Hopkins community will help the PI to set-up collaboration and learn new techniques to build an independent research career.